Betaine and Total Dietary Fiber Recovery from Red Beetroot Peels by Means of Ultrasound and Pulsed Electric Fields

Abstract

Ultrasound-assisted extraction (UAE) using water as a green solvent is a promising non-thermal technique for the extraction of total dietary fiber (TDF) and betaine from red beetroot (Beta vulgaris L.) peel. Compared to conventional thermal extraction (CE), UAE has proven to be a more efficient alternative method for the extraction of TDF and betaine. The pretreatment of beet was carried out using pulsed electric field (PEF) technology, with the specific energy of the PEF treatment set at 1.6 kJ/kg. To achieve the maximum betaine concentration of 24.80 µg/mL, the optimum UAE parameters were 50% amplitude with an extraction time of 3 min using distilled water as extraction solvent. The optimum TDF yield of 44.07% was achieved at 75% amplitude, 6 min treatment time and 50% ethanol solution as extraction solvent. These conditions can effectively supplement UAE, especially in the extraction of bioactive compounds from red beetroot peel. However, the TDF obtained in the residue must be evaporated for further use, which increases energy consumption. Ethanol concentration had no statistically significant effect (p > 0.05) on the TDF results, suggesting that distilled water could replace ethanol as a solvent in UAE. This substitution offers environmental and economic advantages, as water is more environmentally friendly and less expensive than ethanol. In addition, the use of distilled water eliminates the need to evaporate ethanol, which is particularly advantageous when the extracted material is intended for fortification or improvement of the technological and functional properties of food products.

1. Introduction

The Food and Agriculture Organization (FAO) has published that around a third of global food production is wasted every year, which corresponds to around 1.3 billion tons of food. In Europe, around 90 million tons of food waste is produced annually, which is the equivalent of 170 million tons of CO₂ released into the environment every year. The food industry is responsible for 60% of total food losses during production, distribution and sales [1,2,3].

One of the industries with the highest percentage of waste and by-products is the fruit and vegetable processing industry. The final product is often less than 50% of the source material and the processing of this material results in a devastating balance of waste and final product. For this reason, there is a growing need to convert fruit and vegetable by-products into commercially valuable products, especially in the form of bioactive compounds and fiber. Sustainable management of fruit and vegetable waste is, therefore, essential, as is the development of new techniques that enable efficient recycling and reuse for value-added food production [2].

For years, red beetroots were mostly consumed as a vegetable (in about 90% of cases) and only minimally processed [4]. Due to numerous findings on the beneficial composition of red beetroot and the possibility of using not only the pulp, but also other associated parts and its individual components, red beetroot has recently been used in the development and production of various foods with positive effects on the human body. The processing of red beetroot produces sufficient by-products and waste, consisting of peel, pulp, pomace, leaves and stems, depending on the situation. This means that in addition to a significant increase in red beetroot production, the production of processing waste and by-products also increases considerably. Igual et al. [5] reported in their study that around 40% of red beetroot is generated as waste during the extraction of liquefied beet for pulping. A Croatian producer of fruit and vegetable products processes about 1000 tons of red beetroot annually, with about 35% of the waste generated during the process (A. Samardžija, personal communication, 30 March 2025). Certain by-products of red beetroot processing, such as its peel, have significant potential for recycling and reuse to enrich certain food products. The peel is a by-product of any red beetroot processing process (especially minimal processing), although it has a good nutritional composition. Red beetroot peel contains around 2–33% fiber, 4–18% protein and 10–12% minerals (depending on the extraction process), with potassium being the most abundant mineral. Recently, its antioxidant and general bioactive properties have been increasingly studied, as it is considered rich in polyphenols and antioxidants [4,6]. Red beetroot is a rich source of bioactive compounds, including betalains (betacyanin and betaxanthin), flavonoids (such as rutin, astragalin, kaempferol and quercetin), terpenoids, saponins, vitamins, phenolic acids (such as gallic acid, p-coumaric acid and caffeic acid), steroids, alkaloids, tannins, dietary fiber, and sugars [7]. Red beetroot is characterized by high concentrations of nitrates and nitrites, with an average of 1379 mg/kg, making it the highest value among root vegetables. These compounds play an important role in supporting respiratory and cardiovascular health, highlighting the potential benefits of red beetroot and its supplements for improving these physiological systems [8].

Dietary fiber is an essential part of an adequate and balanced diet and plays an important role in maintaining overall health, as the benefits of consuming food rich in dietary fiber are numerous and varied. One of the most well-known benefits of dietary fiber is its role in promoting digestive health. Other benefits include promoting gut health, aiding weight control and chronic disease prevention, and improving blood sugar control [7].

Betaine plays several roles in mammalian physiology, which include three main functions. First, as an organic osmolyte, betaine plays a crucial role in maintaining normal cellular volume under osmotic stress conditions that may arise from various environmental or physiological factors. This function is essential for cellular homeostasis and the proper functioning of cells in response to fluctuating osmotic pressures. Secondly, betaine provides protection against protein denaturation, which is an essential aspect of maintaining cellular integrity and function, especially under stressful conditions such as heat shock or oxidative stress. Thirdly, betaine is significantly involved in the remethylation of homocysteine to methionine as part of the one-carbon metabolism. Together with methylfolate, betaine is one of the few compounds that can donate methyl groups to this important biochemical process, emphasizing its importance in maintaining metabolic balance and reducing the risk of homocysteine-related cardiovascular problems. Although betaine is not classified as an essential nutrient due to its ability to be endogenously synthesized from free choline via the enzyme choline dehydrogenase, the body’s ability to produce sufficient amounts of betaine is often inadequate to meet the body’s daily requirements. Consequently, dietary betaine intake is necessary to ensure proper physiological function. Foods rich in betaine or its precursor choline serve as the main sources for the body’s betaine pool. In particular, cereal grains, pseudocereals (especially amaranth and quinoa), whole grain products (e.g., whole wheat flour, bread, pasta, couscous and breakfast cereals) and certain vegetables such as spinach and red beetroot contribute significantly to the absorption of betaine. In addition, betaine is often found in various shellfish, including mussels, oysters, clams and scallops [9]. This paper focuses on two bioactive components found in beetroot, the previously mentioned dietary fiber and betaine.

The development of innovative food processing methods can increase the competitiveness in the food industry market by improving product quality, introducing new products to the market, and reducing production costs [10].

The effects of reversible and irreversible electromechanical destruction of the cell membrane have numerous applications in the food industry, mainly because they are not thought to alter the sensory properties of food or denature proteins and most enzymes. The pulsed electric field (PEF) is often used to improve the extraction yield of desired components from various natural substances. A review of the existing literature shows that the extraction of sugar from sugar beet, betalains from red beetroot, inulin from chicory, anthocyanins from red cabbage, polyphenols from fresh tea leaves and many others has been successful. PEF technology is based on the electrical treatment of the sample in an extremely short time (from a few nanoseconds to a few milliseconds), with the strength of the electric field pulses typically ranging between 15–80 kV/cm. The treatment of food with PEF leads to a non-thermal electroplasmolysis of the cell contents, which breaks down the cell membrane [11].

The most common extraction methods are still conventional techniques such as maceration and digestion (a process similar to maceration in which slight heating is applied during extraction), but these have numerous disadvantages due to the nature of the process. Conventional extraction methods require the use of large volumes of organic solvents, are time-consuming and energy consuming, and yield lower quantities of the desired product. They also often have a toxic effect on the environment. To overcome the limitations of conventional methods, environmentally friendly green extraction techniques are currently being developed. These techniques require less use of solvents or the use of “green” solvents, lower process temperatures and shorter extraction times. These improvements, combined with optimized process parameters, result in lower energy consumption, higher yields and less environmental impact. Ultrasonic-assisted extraction, a non-thermal process in which acoustic energy is used to increase the release and diffusion rates of the target substances by cavitation of the solvent, is one of these environmentally friendly extraction techniques. The impact of ultrasonic wave-induced acoustic cavitation on the treated matrix contributes significantly to the disruption of cell walls. This process improves both mass and heat transfer, allowing the desired components to diffuse more easily into the extraction solvent, ultimately leading to a higher extraction yield. Ultrasonic waves and their effects alter the structure of the cellular material by causing fragmentation, erosion, tissue tension and dexturization while stimulating redox reactions in the system. Numerous studies have been conducted on the efficacy of ultrasonic extraction, especially of plant metabolites due to the above mechanisms, such as the extraction of chlorophyll from spinach, polyphenols from apple pomace, betalain antioxidants from beet pomace, oil from caraway seeds and oil from fresh yeast cells [3,12,13].

This study focuses on sustainability by utilizing industrial waste materials as a source of betaine and dietary fiber, combined with nonthermal techniques that minimize environmental impact. The pretreatment of beetroot was carried out using PEF technology. PEF pretreatment was used to facilitate the peeling of beets, thereby reducing waste, and maximizing the utilization of by-products from red beetroot processing. The efficacy of ultrasound-assisted extraction (UAE) and conventional thermal extraction (CE) of betaine and dietary fiber from beetroot peel was evaluated by varying the applied amplitude, solvent, and treatment time for UAE extraction, as well as the applied solvent and treatment time for CE extraction. To our current knowledge, there is no reference to the UAE extraction of betaine from beetroot peel. The aim was to obtain a high amount of betaine and dietary fiber, which could then be used as a source of bioactive components for functional food production and upcycling of red beetroot peel.

2. Materials and Methods

2.1. Plant Materials

Red beetroot (Beta vulgaris L.) was provided by Naturala d.o.o., Slatina, Croatia. PEF pretreatment (PEF Advantage Belt, Elea Technology GmbH, Quakenbrück, Germany) of red beetroot was carried out at Kanaan d.o.o., Croatia. The specific energy of the PEF pre-treatment was 1.6 kJ/kg. Red beetroot was manually peeled. To facilitate extraction during sample preparation, the peels were ground with a cryogenic mill (Stephan Universal Machine UMC 5, Stephan Food Service Equipment GmbH, Hameln, Germany) for 2 min at speeds 1, 2 and 3. A second grinding was carried out with Retsch Grindomix GM200 (Retsch GmBH, Haan, Germany) with the programs HIT (4000 rpm, 20 s) and CUT (2000 rpm, 30 s). The samples were stored in a freezer at −20 °C (Gorenje GSI d.o.o., Ljubljana, Slovenia) until the start of the analysis.

2.2. Labelling of the Samples and Extraction

2.2.1. Sample Labels

For both extraction methods, UAE and CE samples were prepared and labeled as shown in

Table 1. Numerical labels and process parameters for UAE-treated samples.

Ethanol Content (%)	UAE-Treated	Parameter Number	Amplitude (%)	Treatment Time (min)
		1	75	3
		2	100	9
		3	50	3
0		4	100	3
25	CU	5	50	9
50		6	50	6
		7	75	6
		8	100	6
		9	75	9

and

Table 2. Numerical labels and process parameters for CE-treated samples.

Ethanol Content (%)	CE-Treated	Parameter Number	Treatment Time (min)
0		1	3
25	CK	2	6
50		3	9

. The numerical labels were randomly assigned to the samples during experimental design using the Statgraphics Centurion program (StatPoint Technologies, Inc., Warrenton, VA, USA). Each number indicates different values for the parameters: treatment time, solvent and amplitude for UAE-extracted samples or treatment time and solvent for CE-extracted samples. For example, sample 0CU4 represents a sample that was UAE-treated with 0% ethanol (distilled water) as an extraction solvent and with an amplitude of 50% over a treatment time of 3 min.

2.2.2. Chemicals

Most chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemicals from other producers are listed below: Celite (Neogen, MI, USA), distilled and deionized water (Eurofins Croatiakontrola d.o.o., Zagreb, Croatia), MES/TRIS buffer 0.05 M (Neogen, MI, USA), Megazyme TDF Assay Kit (Neogen, MI, USA), Acetonitrile ≥ 99.9% for LC-MS (J. T. Baker, Avantor Performance Materials Poland S. A., Gliwice, Poland), Ammonium formate, ≥99.0% for HPLC (Honeywell FlukaTM, Germany), Formic acid, ca. 98%, for mass spectrometry (Honeywell FlukaTM, Germany).

2.2.3. Ultrasound-Assisted Extraction (UAE)

All UAE were performed using Q700CA Sonicator (Qsonica, CT, USA, 20 kHz) by adding 100 mL solvent (distilled water, 25% ethanol and 50% ethanol) into a 250 mL laboratory beaker with 5 ± 0.0001 g of weighed sample. The sample mass and solvent volume were determined based on previous experience, and this ratio has been shown to be effective for extraction in other samples. This ratio of sample mass to solvent volume prevents the sample from adhering to the ultrasonic probe. During extraction, an ultrasound probe (diameter 12 mm) was placed in the center of the laboratory beaker and immersed into the liquid at about a 2.4 cm depth, sufficiently spaced from the bottom. In addition, all UAE treatments were performed according to the previously optimized extraction parameters: amplitude of 50%, 75%, and 100%, and treatment time of 3, 6, and 9 min. To avoid overheating, the laboratory beakers were placed in a plastic container with ice cubes and water. The mean value of the effective ultrasonic power of the process was 75 W for all samples treated with distilled water and for samples treated with a mixture of ethanol and water as a solvent, it was 90 W. The parameters for this study were determined using a three-level multifactor design approach supported by the Statgraphics Centurion program (StatPoint Technologies, Inc., Warrenton, VA, USA). The initial values for the parameters were derived from preliminary tests performed on other samples. The extraction parameters as a function of the sample and the solvent used are listed in Table 1. The extraction parameters, depending on the sample and solvent used, can be found in Table 1. The UAE of each sample was performed in four replicates to obtain sufficient amounts of extract and extraction residues for betaine and TDF analysis. At the end of the extraction, the samples were filtered through a Büchner funnel, which was placed on a suction bottle and connected to a vacuum pump. Total dietary fiber was determined in the extraction residue, which remained on the filter, and betaine was determined in the extract.

During the extraction of a single sample, the temperature, power and energy values displayed on the control LCD monitor were recorded every 15 s. The total energy change [W] in a given time interval was calculated using the following equation:

$$P_t=m\times c_p\times {\displaystyle \frac{dT}{t}}$$

(1)

where P_t is the total power change [W], m is the mass of the treated sample (beet peel and solvent) [g], c_p is the specific heat capacity of the beet peel [J/g°C], dT is the temperature change in an interval of 15 s [°C] and t is the time [s].

$$c_p=x_w\times c_{p,w}+x_{carbo}{\times c}_{p,carbo}+x_p{\times c}_{p,p}+x_f\times c_{p,f}+x_a\times c_{p,a}$$

(2)

where c_p is the specific heat capacity of the beet peel [J/g°C], x_n is the proportion of the individual component of the beet peel (water, carbohydrates, proteins, fats, ash), and c_p,n is the specific heat capacity of the individual component (water, carbohydrates, proteins, fats, ash) [J/g°C].

Using the calculated values of the total energy change, the values of power density [W/cm³] with respect to the volume of the treated sample (100 mL) and power density [W/cm²] with respect to the area of the tip of the ultrasonic probe (113.0973 mm²) were obtained.

2.2.4. Conventional Thermal Extraction (CE)

All CE treatments were performed in a water bath (Inkolab d.o.o, Zagreb, Croatia) by adding 100 mL of solvent (distilled water, 25% ethanol and 50% ethanol) to a 250 mL laboratory beaker containing 5 ± 0.0001 g of weighed sample. In addition, all CE treatments were performed according to the previously optimized extraction parameters: treatment time of 3, 6, and 9 min and temperature of 60 °C. The extraction parameters depending on the sample and the solvent used are listed in Table 2.

The CE of each individual sample was performed in four replicates to obtain sufficient amounts of extract and extraction residues for betaine and TDF analysis. At the end of the extraction, the samples were filtered through a Büchner funnel placed on a suction bottle and connected to a vacuum pump. Total dietary fiber was determined in the extraction residue, which remained on the filter, and betaine was determined in the extract.

2.3. Analysis

2.3.1. Determination of Total Dietary Fiber Content (TDF)

The samples were dried for 16 h at 103 °C in a laboratory oven (Inkolab d.o.o, Zagreb, Croatia) before analysis. TDF content was determined according to the method AOAC 991.43 [14] by treating the sample with thermostable α-amylase to gelatinize it, protease to remove proteins and amylglucosidase to remove starch. The soluble fiber was precipitated by adding ethanol. The sample was then filtered and washed first with ethanol and then with acetone. The residue on the crucible filter was dried and weighed. Two parallel determinations were carried out, one to determine the protein content and the other to determine the ash content. The proteins were determined using the Kjeldahl method [15]. The ash was determined in a muffle furnace (Inkolab d.o.o., Zagreb, Croatia) by incinerating the samples at 550 °C for 5 h [16]. The TDF content in each sample was determined in parallel.

The TDF content was calculated by correcting the protein and ash values obtained and the blank test performed during the determination of each sample.

TDF content was calculated according to the equation:

$$TDF={\displaystyle \frac{m_{ou}-B-P-S}{m}}\times FP\times 100$$

(3)

where TDF is percentage of total dietary fiber [%], m_ou is mean value of the mass residue for two sample determinations [mg], B is mass of the residual protein determined in one sample residue [mg], P is mass of the ash determined in second sample residue [mg], S is blank value [mg], m is mean value of the mass of samples weighted for both parallels [mg] and FP is conversion factor.

2.3.2. Determination of Betaine Content

The sample extract was prepared according to the method published by Rivoira et al. (2017) with a few modifications [17]. The betaine content in each sample was determined in parallel.

Sample Extract Preparation

Sample preparation was carried out according to the “dilute and shoot” principle. The extracts were filtered with a nylon syringe filter with a pore diameter of 0.22 µm and diluted 1000 times with water.

Betaine Standard Solutions

Betaine stock solution was prepared by dissolving the appropriate amount of 0.0555 g of the pure standard (betaine, 99.2%, Sigma-Aldrich, St. Louis, MO, USA) in deionized water. The prepared standard solution had a concentration of 1.1011 mg/mL. The working and calibration solutions were prepared by diluting the stock solution with water.

Preparation of Betaine Calibration Solutions

Betaine calibration solutions for the preparation of the baseline were prepared from the intermediate standard solution. Precise volumes of the intermediate solution were pipetted into special vials to prepare the calibration solutions. This method ensures that the calibration points are within the linear range of the calibration curve. The mass concentration of each calibration solution was determined by dividing the concentration of the solution used by the dilution factor.

Preparation of the Mobile Phase

Ammonium formate (0.3154 g) was weighed into a 500 mL laboratory beaker and 300 mL deionized water was added. The beaker was placed in an ultrasonic bath (Sonorex Digitec DT510H, BANDELIN electronic GmbH & Co. KG, Berlin, Germany) for 1 min to completely dissolve the ammonium formate. After dissolution, the beaker was placed on a magnetic stirrer (IKA Plate RTC digital, IKA Werke GmbH & Co., Staufen im Breisgau, Germany) and formic acid was added to the mixture to adjust the pH to 3. The initial pH of the mixture was 6.26, and the final pH was 3.03, which was achieved by adding 790 µL of formic acid. The solution was then topped up to the 500 mL mark with deionized water. The mobile phase was prepared using a mixture of ammonium formate solution and acetonitrile, which was chosen for its ability to ensure optimal separation and ionization of the analytes. The aqueous phase consisted of a 25% ammonium formate solution (A), while the organic phase consisted of 75% acetonitrile (B).

LC-MS/MS Analysis

High-performance liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) was used for the determination of betaine in red beetroot peel extracts. The HPLC system (Agilent Technologies, CA, USA) included the following components: degasser (Model G1379B Agilent 1260 Infinity Micro Degasser), binary pump (Model G1312B Agilent 1260 Infinity Binary Pump), high pressure autosampler (Model G1367E Agilent 1260 Infinity High Performance Autosampler) with thermostat (G1330B Agilent 1290 Infinity Thermostat) and thermostatic column compartment (Model G1316C Agilent 1290 Infinity Thermostated Column Compartment) in conjunction with a triple quadrupole (QQQ) mass spectrometer (6460 Triple Quad LC/MS, Agilent Technologies, CA, USA) equipped with a jetstream electrospray ionization (ESI) source. The separation was performed using a Kinetex^® 2.6 µm HILIC 100 Å column (Phenomenex, CA, USA) with the dimensions 100 × 4.6 mm. The temperature of the column was 40 °C and the temperature of the autosampler temperature was set to 10 °C. The mobile phase consisted of (A) 0.01 M ammonium buffer pH 3 and (B) acetonitrile. The flow rate of the mobile phase was 0.8 mL/min and the injection volume was 2 µL. The elution was isocratic with a ratio of 25:75.

The betaine molecules were ionized using positive electrospray ionization in a nitrogen ionization chamber at 275 °C with a gas flow of 10 L/min, a nebulizer pressure of 35 psi, a sheath gas temperature of 400 °C, a sheath gas flow of 11 L/min and a capillary voltage of 2500 V, generating a precursor ion (molecular cation) with m/z 118/09. The precursor ion was introduced into the collision cell, where it fragmented upon collision with nitrogen molecules. The fragmentation voltage was set to 108 V. Two product ions were monitored, one with m/z 74.1 (for quantification) and one with m/z 58.1 (for identification). The collision energies for the formation of the ions with m/z 74.1 and 58.1 were 24 V and 32 V, respectively. Quantitative data analysis was performed using the MassHunter program. Multiple Reaction Monitoring (MRM) mode was used for the quantification and identification of betaine, which allows the system to monitor two transitions of the precursor ion [18].

2.3.3. CO₂ Calculation

The CO₂ emission values were calculated based on energy consumption with the electricity/heat factor (0.3414155 kg CO₂ (kWh)⁻¹) determined by the International Energy Agency (IAE) for Croatia.

2.3.4. Experimental Design and Statistical Analysis of UAE and CE

Experimental design and statistical analysis of the extraction parameters of UAE and CE were performed using STATGRAPHICS Centurion (StatPoint Technologies, Inc., Warrenton, VA, USA). The trial comprised 27 UAE samples (9 samples treated with distilled water, 9 samples treated with 25% ethanol and 9 samples treated with 50% ethanol) and 9 CE samples (3 samples treated with distilled water, 3 samples treated with 25% ethanol and 3 samples treated with 50% ethanol). Multilevel Factorial Design was used to determine the potential impact of the input variables (independent) on the output variables (dependent). The independent parameters of the experiment for the UAE-treated samples were: amplitude (50, 75 and 100%), treatment time (3, 6 and 9 min) and solvent (distilled water, 25% ethanol, 50% ethanol). Independent parameters were used for CE-treated samples: treatment time (3, 6 and 9 min) and solvent (distilled water, 25% ethanol, 50% ethanol). For the UAE- and CE-treated samples, total dietary fiber content [%] and betaine content [µg/mL] were the dependent variables. The STATGRAPHICS Centurion program also performed a multivariate analysis of variance (MANOVA) for each output variable, which considers the interactions between two input parameters and the quadratic interaction of each input parameter and tests whether individual output values of the tested properties are affected. The parameters had a statistically significant effect if p < 0.05, meaning that they were significantly different from zero at the 95.0% confidence interval. Furthermore, the optimal TDF and betaine yields were determined via statistical analysis of the data (Response Optimized Model). ANOVA is used for statistical data processing exclusively for CO₂ emissions from conventional extraction (Microsoft Office 365 MSO, Version 2402).

3. Results

3.1. Physical Parameters After UAE and CE Treatment

Total power, energy, and calculated CO₂ emissions for the samples after UAE treatment are shown in Figure 1. The lowest mean values of total power and energy were observed for sample 25CU3, which was treated under the mildest conditions (3 min, amplitude 50%). As CO₂ emissions were calculated from energy, CO₂ emissions were also lowest for sample 25CU3 (7.09 g CO₂). In contrast, the highest values for total power, energy and CO₂ emissions were obtained for sample 50CU2, which was treated under the most intensive conditions (9 min, amplitude 100%). CO₂ emissions for sample 50CU2 were 112.1 g CO₂. The strength of the ultrasound depends on the amplitude and treatment time [19], which explains these results.

In agreement with the results obtained with distilled water as solvent, extraction with 25% ethanol also showed the lowest mean values for total power, energy and CO₂ emissions when performed under mild conditions (25CU3, at 3 min, amplitude 50%). The highest values were again found when the sample was treated under the most intense conditions (25CU2, 9 min and amplitude 100%). When 50% ethanol was used as a solvent, the lowest mean values for total power, energy and CO₂ emissions were again found for the sample treated under mild conditions (50CU3, 3 min, amplitude 50%), while the highest values were observed for the sample treated under the most intensive conditions (50CU2, 9 min, amplitude 100%).

The observed results can be explained by two key factors: (i) the temperature increase with longer treatment time and (ii) the specific heat capacity of ethanol is about half that of water (cp(H₂O,l) = 4.184 J g⁻¹ K⁻¹, cp(ethanol,l) = 2.46 J g⁻¹ K⁻¹), which resulted in ethanol reaching a higher temperature than water for the same treatment time (NIST Chemistry WebBook). Furthermore, only the proportion of ethanol in the solvent affects the total energy change. This can be attributed to the fact that the total energy change is directly related to the temperature change in a given time interval. Since ethanol experiences a greater temperature increase than pure water over the same period of time, the resulting temperature differences lead to a greater influence of the solvent on the total energy change. The CO₂ emission values for the extraction were calculated based on the energy consumption, so that everything relates to CO₂. For each extraction, the emissions of gCO₂ per gram of TDF and gCO₂ per milligram of betaine were calculated. For the UAE extractions, CO₂ emissions ranged from 277.1 to 3432.29 gCO₂/g TDF, while betaine emissions varied from 292.47 to 5297.26 gCO₂/mg betaine.

The energy and calculated CO₂ emissions for the samples after CE treatment are shown in

Table 3. Data obtained via CE for samples: Energy and CO₂ emissions.

Treatment Time (min)	Energy (J)	CO2 (g)
3	684,000	64.87
6	1,404,000	133.16
9	2,088,000	198.04

. The lowest energy values and CO₂ emissions were determined for the samples subjected to the shortest treatment time (3 min). In contrast, the highest energy values and CO₂ emissions were determined for the samples with the longest treatment time (9 min). For the CE extractions, CO₂ emissions ranged from 2097.45 to 18,942.06 gCO₂/g TDF, while betaine emissions ranged from 2771.62 to 15,147.66 gCO₂/mg betaine.

3.2. Total Dietary Fibre (TDF) Content

Comparison of TDF Yields Achieved by UAE and CE

The results in Figure 2 show that the percentage of TDF extracted via ultrasound ranges from 25% to 63.55%. In this study, it was observed that the TDF content in the extraction residue can increase with increasing treatment time, amplitude, and ethanol concentration in the solvent. This indicates that an increase in these parameters leads to a lower extraction yield in the extract and a higher extraction yield in the extraction residue.

The optimum TDF yield (44.07%) was obtained at an amplitude of 75% amplitude, a treatment time of 6 min and a 50% ethanol solution as solvent. UAE is expected to result in a higher yield of TDF in the solvent due to the physicochemical effects it exerts on the treated medium. This would normally lead to a lower proportion of dietary fiber in the peel residue. However, this assumption is not correct in the present situation. Ethanol as a solvent has a coagulating effect on the fibers, which results in the TDF remaining in the extraction residue and not being extracted into the solvent. It is important to consider the temperature conditions during the extraction process. The average maximum temperatures reached during ultrasonic extraction were 33.4 °C, 37.9 °C and 40 °C for 0%, 25% and 50% ethanol, respectively. In contrast, conventional thermal extractions were performed at a constant temperature of 60 °C, which is at least 1.5 times higher than the highest temperatures reached during ultrasonic extraction. This temperature difference can significantly affect the extraction dynamics and emphasizes the need to carefully control the thermal conditions during ultrasonic extraction.

For the samples treated with UAE, mutual interaction and the individual quadratic interactions of amplitude, treatment time and solvent showed no statistically significant effect on the yield of TDF (p > 0.05), as shown in

Table 4. Statistical significance for TDF yield. MANOVA statistically processes the variability of each input parameter, their mutual interactions, and quadratic interactions of treatment time, amplitude, and solution (UAE).

Sample	Main Effects			Interactions
	A: Amplitude	B: Treatment time	C: %EtOH	AA	AB	AC	BB	BC	CC
p	0.85	0.41	0.17	0.07	0.92	0.81	0.32	0.92	0.56

.

The results in Figure 2 show that the proportion of TDF extracted via conventional thermal extraction ranges from 7.03% to 35.11%. For the CE-treated samples, mutual interaction and the individual quadratic interactions of treatment time and solvent showed no statistically significant effect on the yield of TDF (p > 0.05), as shown in

Table 5. Statistical significance for TDF yield. MANOVA statistically processes the variability of each input parameter, their mutual interactions, and quadratic interactions of treatment time and solution (CE).

Sample	Main Effects		Interactions
	A: Treatment time	B: %EtOH	AA	AB	BB
p	0.41	0.17	0.07	0.92	0.81

.

3.3. Betaine Content

Comparison of Betaine Yields Achieved by UAE and CE

The betaine results obtained from UAE are presented in Figure 3. Using distilled water as a solvent, it can be observed that with a short treatment time (3 min), an amplitude of 50% gives the best result (0CU3), while increasing the amplitude to 100% does not increase the betaine yield (0CU4). For the medium treatment time (6 min), a similar trend can be observed as for the shorter time—an amplitude of 50% leads to the best results (0CU6), while increasing the amplitude to 75% (0CU7) or 100% (0CU8) leads to a lower betaine yield. With a longer treatment time (9 min), the differences between the different amplitudes are less pronounced, but an amplitude of 50% (0CU5) again showed slightly better results than an amplitude of 100% (0CU2). The combined effect of amplitude and treatment time showed that the highest betaine yield in the samples treated with ultrasound was achieved at an amplitude of 50%, especially at shorter and medium treatment times (3 and 6 min). Increasing the amplitude to 100% generally reduced the betaine yield, while longer treatment times (9 min) may attenuate this effect, but not enough to outperform the lower amplitude results.

When using a 25% ethanol as solvent, it can be observed that with a shorter treatment time of 3 min, an amplitude of 100% gave the highest betaine yield (25CU4), while reducing the amplitude to 50% resulted in a lower yield (25CU3). However, the differences are relatively small. At the medium treatment time of 6 min, an amplitude of 50% resulted in a higher betaine yield (25CU6) than higher amplitudes of 75% and 100% (25CU7, 25CU8). With a longer treatment time of 9 min, an amplitude of 100% again resulted in the highest betaine yield (25CU2), while lower amplitudes of 50% led to a lower yield (25CU5). The combined effect of amplitude and treatment time showed that the highest betaine yield was found in the samples treated with an amplitude of 100%, especially at shorter and longer treatment times—3 and 9 min. However, for the medium treatment time of 6 min, a lower amplitude of 50% showed slightly better results. When using 50% ethanol as a solvent, it can be observed that at a short treatment time of 3 min and higher amplitude of 100% gave the highest betaine yield (50CU4), while a decrease in amplitude resulted in a lower yield. At the medium treatment time of 6 min, an amplitude of 75% resulted in the highest betaine yield (50CU7), while the results for amplitudes of 50% and 100% were very similar and slightly lower (50CU6, 50CU8). With a longer treatment time of 9 min, a higher amplitude of 100% again led to the highest betaine yield (50CU2), while a reduction in amplitude led to a lower yield. The combination of a higher amplitude of 100% with shorter—3 min—or longer—9 min—treatment times resulted in a higher betaine yield (50CU4, 50CU2), indicating that an amplitude of 100% was optimal for betaine extraction under these conditions (50CU2). On the other hand, an amplitude of 75% led to the best results at the medium treatment time of 6 min (50CU7), but in general, higher amplitudes proved to be more effective. The 50% amplitude consistently produced the lowest concentrations, indicating that lower amplitudes are not optimal for betaine extraction under these conditions. The highest betaine yield was measured in sample 0CU3 with a value of 25.30 µg/mL, while the lowest betaine yield was measured in sample 0CU1 with a value of 19.91 µg/mL. There is no clear trend indicating that a higher ethanol content increases or decreases the betaine yield. The betaine yield varied with different ethanol content, but other amplitudes and times also influenced the result. Thus, although ethanol may have some effect on betaine yield, changes in amplitude and time were also critical, and their combined effect was important in determining the final betaine content in the samples. The optimum betaine yield (24.80 µg/mL) was achieved at an amplitude of 50%, a treatment time of 3 min and distilled water as solvent.

For the samples treated with UAE, most of the mutual interactions and single quadratic interactions of amplitude, treatment time and solvent showed no statistically significant effect on the yield of betaine (p > 0.05), as shown in

Table 6. Statistical significance for betaine yield. MANOVA statistically processes the variability of each input parameter, their mutual interactions, and quadratic interactions of treatment time and solution (UAE).

Sample	Main Effects			Interactions
	A: Amplitude	B: Treatment time	C: %EtOH	AA	AB	AC	BB	BC	CC
p	0.31	0.07	0.89	0.48	0.47	0.26	0.74	0.01	0.86

. A statistically significant influence of the mutual interaction of treatment time and solvent was recorded (p < 0.05).

The betaine results of the CE are presented in Figure 3. For the samples with distilled water, there is a slight decrease in the betaine content with increasing time (3, 6, 9 min). For example, 0CK1 (3 min, distilled water) has the highest betaine content, 23.41 µg/mL, while 0CK3 (9 min, distilled water) has a lower content, 20.53 µg/mL. For the samples with different ethanol contents (25% or 50%), the effect of time is not as pronounced, and it could be seen that the influence of the ethanol content is more significant. For samples with a 25% ethanol, the betaine content was generally higher than at 50% ethanol, but lower than with distilled water. For example, 25CK1 (3 min, 25% ethanol) has a betaine content of 20.12 µg/mL, while 50CK1 (3 min, 50% ethanol) has a betaine content of 18.84 µg/mL. The betaine concentration generally decreased with increasing ethanol content, which can be explained by the better solubility of betaine in water than in ethanol [20]. In addition, the betaine content generally decreased with increasing treatment duration.

For the CE-treated samples, most of the mutual interactions and individual quadratic interactions of amplitude, treatment time and solvent showed no statistically significant effect on the yield of betaine (p > 0.05), as shown in

Table 7. Statistical significance for betaine yield. MANOVA statistically processes the variability of each input parameter, their mutual interactions, and quadratic interactions of treatment time and solution (CE).

Sample	Main Effects		Intercations
	A: Treatment time	B: %EtOH	AA	AB	BB
p	0.40	0.43	0.06	0.01	0.93

. A statistically significant influence of the mutual interaction of treatment time and solvent was recorded (p < 0.05).

4. Discussion

The application of non-thermal extraction techniques, together with the use of environmentally friendly solvents, plays an important role in reducing costs and promoting an environmentally friendly approach compared to conventional extraction methods. The pretreatment of red beetroot was carried out using PEF technology to facilitate manual peeling. The proportion of red beetroot peel pre-treated with PEF was 11%, compared to a process without PEF pre-treatment, where the proportion of peel is 35%. The results of this study are consistent with the results of Giancaterino and Jaeger [21], who investigated the effects of pulsed PEF treatment on the peeling effect of tomatoes and kiwis. Their research showed that PEF treatment significantly improved the peeling efficiency of both fruits, confirming the results of our own study. In the work of Giancaterino and Jaeger [21], PEF treatment led to a reduction in peeling loss in tomatoes from 43% to 33%, demonstrating the potential of PEF to improve the efficiency of the peeling process. This reduction in peel loss suggests that PEF treatment may improve peel detachment from the fruit, likely due to the breakdown of cell wall structures and weakening of intercellular bonds, a mechanism often associated with the effects of PEF on plant tissue. In our study, an increase in the peeling effect was also observed, although the exact extent of the improvement may vary due to different experimental conditions, such as the specific PEF parameters used and the type of fruit. However, the benefits of PEF treatment were even more pronounced in kiwifruit. Giancaterino and Jaeger [21] reported up to 66% less peel loss in the PEF-treated kiwifruit compared to the untreated samples, indicating a significant improvement in peeling efficiency. This result is particularly noteworthy, as kiwifruit has a relatively thick, fuzzy skin that is more difficult to remove compared to other fruits. The effectiveness of PEF in reducing peel loss in kiwifruit may indicate that PEF treatment is particularly beneficial for fruit with complex or tough peel structures. In addition, the evaluation methods used by Giancaterino and Jaeger [21], which include both manual and mechanical peeling as well as the evaluation of weight loss during the peeling process, provide a comprehensive understanding of the effects of PEF treatment. Weight loss during peeling can be a critical factor in food processing as it has a direct impact on yield and the economic profitability of the process. The reduction in peel loss observed in both tomatoes and kiwifruit could, therefore, have important implications for the food industry, particularly in terms of improving fruit processing efficiency while minimizing waste.

As part of the research, samples of red beetroot peel were subjected to a UAE and a CE extraction to determine and optimize the yield of total dietary fiber in the extraction residue and betaine in the extract. UAE was used due to the possibility of achieving relatively low treatment temperatures, while the selected extraction solvents, distilled water and ethanol, represent a cheap, ecologically and technologically acceptable “green solvent” [19,22,23].

In general, ultrasonically treated samples were found to have higher levels of TDF in the extract residues and betaine in the extracts compared to samples subjected to conventional thermal extraction. The TDF yield ranged from 25.55% to 63.55% in the ultrasonically treated samples. Optimum input values of amplitude, treatment time and ethanol content for the maximum output value of TDF were determined via the Response Optimized Model in STATAGRAFICS Centurion (StatPoint Technologies, Inc., Warrenton, VA, USA). For the maximum output value of 44.07%, the optimal parameters are 76.71% amplitude, 6.59 min and 50% ethanol solution. In general, consistent trends were observed; TDF in the extraction residue increased with treatment time, amplitude, and ethanol concentration in the solvent. Accordingly, an increase in these parameters led to a lower extraction yield in the extract, as the TDF content was determined in the extraction residue and not in the extract. Insoluble dietary fibers are not soluble in water anyway (and usually not in ethanol either), or the ethanol content in combination with other mild processing conditions does not create conditions under which they can be successfully extracted. On the other hand, ethanol causes precipitation of soluble fiber (less polar solvent than water), which prevents its transfer from the plant to the extract and makes permeability more difficult. Both phenomena could explain the abundance of TDF in the extraction residue of samples extracted with ultrasound using a higher ethanol content in the solvent compared to samples extracted under milder parameters (e.g., water), potentially improving mass transfer into the extract [24]. From this point of view, the lack of a statistically significant influence of the ethanol content in the solvent is logical. Regarding the treatment time, it is important to emphasize that compared to previous studies and the optimization of the treatment duration parameter, it was found that the range of the time interval is much smaller. Indeed, most studies show that there is a positive correlation between the extended treatment time and the extraction yield when the treatment time exceeds 10 min, which is longer than the longest treatment time used in this study [25,26]. The optimum yield of plant metabolites, including fibers, is achieved in a time range of 10 to 60 min, after which the efficiency decreases [27]. Therefore, it is possible that the treatment time interval used in this study (with a maximum of 9 min) is too short to achieve a statistically significant effect of the treatment time parameter. The same listed inconsistencies may also be an explanation for the lack of statistical significance of the effect of amplitude on extraction yield, as the trend of yield increase as a function of process duration and applied ultrasonic power are consistent with each other [28]. Results such as our research findings were recorded in the study on the optimization of ultrasound-assisted extraction of dietary fiber from yellow dragon fruit peels, which resulted in the highest insoluble dietary fiber (IDF) (61.3%) and soluble dietary fiber (SDF) (10.8%) values. The best extraction conditions were achieved by maximizing IDF and SDF with the following parameters: pause time 1 s, liquid to solid ratio 30 mL/g and total treatment time 60 min [29]. Another study investigated the potential of extracting dietary fiber from the Queen pineapple of the Tripura region using ultrasound-assisted extraction (UAE). The UAE method was compared with the traditional alkali extraction technique for extracting dietary fiber from the peel waste of the Queen pineapple. The results showed that the UAE method achieved a yield of 86.67% after 22.35 min of sonication, with a solid/liquid ratio of 27.5 g/mL and an ultrasonic amplitude of 46.9% [30].

The total betaine yield ranges from 19.91 µg/mL to 24.37 µg/mL in the samples treated with ultrasound. In the samples treated with UAE, most of the two-way interactions and single quadratic interactions of amplitude, treatment time and solvent showed no statistically significant effect on betaine yield (p > 0.05). A statistically significant influence of the mutual interaction of treatment time and solvent was found (p = 0.01). Optimum input values of amplitude, treatment time and ethanol content for the maximum output value of betaine were determined using the Response Optimized Model in STATAGRAFICS Centurion (StatPoint Technologies, Inc., Warrenton, VA, USA). For the maximum output value of 24.80 µg/mL, the optimal parameters are 100% amplitude, 3 min, and distilled water. For the samples extracted with CE, the betaine yield was lower compared to the samples extracted with UAE. The decrease in betaine yield with conventional extraction (CE) can be attributed to the degradation of betaine due to prolonged exposure to heat. In general, most biologically active compounds are sensitive to heat. We found no data on betaine extraction from red beetroot and its by-products to compare. Most researchers investigated the extraction of betalain and betanin. For example, Aztatzi-Rugerio et al. [31] investigated the betaine concentration at 75 °C over different time intervals and observed the degradation of betanins, a pigment group of betalains, which led to the formation of neobetanin, the main degradation product of betalains. In comparison, the highest average temperatures during ultrasound-assisted extraction (UAE) were 25.25, 28 and 29.3 °C, while CE was performed at a constant temperature of 60 °C, which probably contributed to the greater degradation of betaines. Despite the limited number of studies dealing with betaine extraction, one study presents an efficient and environmentally friendly method for the extraction of betaine from sugar beet waste (molasses) in the sugar industry. The method uses a sustainable surfactant and is based on the technique of cloud point extraction (CPE). Under optimized conditions, the extraction efficiency for betaine recovery reached up to 88%. Polyethylene glycol (PEG), an edible surfactant, was used in the process. In the final step, the extracted betaine was freeze-dried at −56 °C for 16 h under 0.5 bar ambient pressure. The results indicate that the final betaine powder product is suitable for direct use as a dietary supplement in animal nutrition given the safety of PEG for consumption [32]. Borjan et al. [33] compared Soxhlet, ultrasonic, cold and supercritical fluid extraction methods. Soxhlet extraction with water as solvent yielded the highest amount of betalain, while Soxhlet extraction with 50% ethanol as a solvent yielded the highest concentration of total phenols. The total phenol concentration ranged from 12.09 mg/g to 18.60 mg/g. Ultrasonic extraction with a solvent of 30% methanol gave the lowest concentration of total phenols, while Soxhlet extraction with 50% ethanol gave the highest concentration. Cold extraction with 50% methanol showed the highest anti-inflammatory activity. The authors concluded that Soxhlet extraction with 50% ethanol is the most suitable method for the extraction of bioactive compounds, as it has a higher yield and bioactivity. Red beetroot is a valuable source of betalains, natural red pigments, polyphenols, fiber and nitrates, and its popularity (especially in the form of juice) has resulted in a considerable amount of waste being generated. Fernando et al. [3] focused their study on the recovery of betalains and polyphenols from dried whole red beetroot and beet pulp waste from the juicing industry. For UAE, ethanol/water-based solvent mixtures were used, which proved to be more effective than single solvents. Initially, enzyme-assisted extraction was tested for wet beet pulp, but this method failed to obtain the betalains. The results indicate that the betalains are more stable in dried pulp, making it a preferred starting material for extraction. Another study was focused on the UAE of betalains from red beetroot using water as a solvent. The effects of extraction time and temperature and the comparison of UAE with orbital shaking extraction (OSE) were investigated to evaluate the influence of ultrasound on the extraction process. The optimal conditions for UAE were determined and further studies on ultrasonic power and solvent/sample ratio were conducted. The results showed that the betalain content in the extract decreased with increasing temperature, with a similar decrease observed at higher extraction times. Consequently, the optimum conditions were set at 30 °C and 30 min. Under these conditions, UAE outperformed OSE in terms of betalain extraction efficiency. In addition, increasing the ultrasonic power improved the extraction process, with 83 W being sufficient for maximum removal of betalains. The solvent-to-sample ratio also played an important role, with the highest betalain content achieved at a ratio of 75 mL/g, which corresponded to the highest diffusion coefficient under these conditions [34].

This study highlights the importance of the sustainability of extraction processes, particularly regarding the power and energy consumption associated with thermal and ultrasonic extraction techniques. The results show that ultrasonic treatment consumes less energy compared to thermal treatment. As a non-thermal extraction method, ultrasound operates at lower temperatures and significantly reduces the required extraction time, which directly contributes to lower energy consumption [35]. Higher energy consumption is often associated with higher emissions of CO₂ and other pollutants, as reported by [36].

In this study, ultrasonic treatment resulted not only in lower energy consumption but also in a reduction of CO₂ emissions compared to conventional thermal extraction. For the samples treated with ultrasound, the interactions between amplitude, treatment time and solvent showed statistically significant effects on CO₂ emissions (p < 0.05) and for the samples treated with CE, treatment time and solvent showed statistically significant effects on CO₂ emissions (p < 0.05). This double benefit of lower energy consumption and reduced environmental impact makes ultrasound a more sustainable extraction method.

Red beetroot peel is becoming an increasingly popular source of bioactive compounds such as dietary fibers and betaine, which are extracted in a much larger quantity with ultrasound and whose antioxidant properties are better preserved than with CE extraction.

5. Conclusions

Red beetroot peel, which is a by-product from the vegetable processing industry, is a good source of dietary fiber and betaine. In this study, it was shown that dietary fibers and betaine can be extracted in a very efficient and economical way by using green solvents and non-thermal extraction techniques. PEF pretreatment of red beetroot facilitates manual peeling. UAE extraction proved to be more efficient than CE extraction for the extraction of total dietary fiber and betaine. For the samples treated with UAE, single quadratic interactions of amplitude, treatment time and solvent showed no statistically significant effect on the yield of betaine (p > 0.05). Betaine is more soluble in water than in ethanol. Ethanol causes precipitation of the soluble fiber (less polar solvent than water), preventing its transfer from the plant to the extract and leaving it in the extraction residue. Taken together, this suggests that distilled water could replace ethanol as a solvent in the UAE. This substitution offers environmental and economic benefits, as water is more environmentally friendly and less expensive than ethanol. In addition, the use of distilled water eliminates the need to evaporate ethanol, which is particularly advantageous if the extracted material is to be used to fortify or improve the technical and functional properties of food. Considering the nutritional value of dietary fibers and betaine, it is increasingly certain that they will be used in the production of functional products such as cookies, spreads, beverages, etc. For further research, it is essential to scale-up the process to an industrial level to evaluate its feasibility and sustainability under real production conditions. In addition, the health safety of the extraction residues and extract must be investigated to ensure their safety for human consumption.

The development of new products from food waste and by-products has the potential to address global food shortages while reducing environmental impact. This approach not only helps to reduce the amount of food waste, which represents a significant environmental impact, but also contributes to the reduction of greenhouse gas emissions and the conservation of natural resources.